Yaravirus: A novel 80-nm virus infecting Acanthamoeba castellanii Paulo V. M. Borattoa,b,1, Graziele P. Oliveiraa,b,1, Talita B. Machadoa, Ana Cláudia S. P. Andradea, Jean-Pierre Baudoinb,c, Thomas Klosed, Frederik Schulze, Saïd Azzab,c, Philippe Decloquementb,c, Eric Chabrièreb,c, Philippe Colsonb,c, Anthony Levasseurb,c, Bernard La Scolab,c,2, and Jônatas S. Abrahãoa,2 aLaboratório de Vírus, Instituto de Ciências Biológicas, Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil; bMicrobes, Evolution, Phylogeny and Infection, Aix-Marseille Université UM63, Institut de Recherche pour le Développement 198, Assistance Publique–Hôpitaux de Marseille, 13005 Marseille, France; cInstitut Hospitalo-Universitaire Méditerranée Infection, Faculté de Médecine, 13005 Marseille, France; dDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907; and eDepartment of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by James L. Van Etten, University of Nebraska-Lincoln, Lincoln, NE, and approved June 2, 2020 (received for review January 29, 2020) Here we report the discovery of Yaravirus, a lineage of amoebal others. NCDLVs have dsDNA genomes and were proposed to virus with a puzzling origin and evolution. Yaravirus presents share a monophyletic origin based on criteria that include the 80-nm-sized particles and a 44,924-bp dsDNA genome encoding sharing of a set of ancestral vertically inherited genes (17, 18). for 74 predicted proteins. Yaravirus genome annotation showed From this handful set of genes, a core gene cluster is found to be that none of its genes matched with sequences of known organ- present in almost all members of the NCLDVs, being composed isms at the nucleotide level; at the amino acid level, six predicted by five distinct genes, namely a DNA polymerase family B, a proteins had distant matches in the nr database. Complimentary primase-helicase, a packaging ATPase, a transcription factor, and prediction of three-dimensional structures indicated possible func- a major capsid protein (MCP) for which the double jelly-roll tion of 17 proteins in total. Furthermore, we were not able to (DJR) fold constitutes the main protein architectural class (19, retrieve viral genomes closely related to Yaravirus in 8,535 publicly 20). Recently, the International Committee on Taxonomy of available metagenomes spanning diverse habitats around the globe. Viruses (ICTV) brought forward a proposal for megataxonomy of The Yaravirus genome also contained six types of tRNAs that did not viruses (21). The DJR major capsid protein (MCP) supermodule match commonly used codons. Proteomics revealed that Yaravirus of DNA viruses includes NCLDVs and other icosahedral viruses MICROBIOLOGY particles contain 26 viral proteins, one of which potentially represent- that infect prokaryotes and eukaryotes. In addition to the signa- ing a divergent major capsid protein (MCP) with a predicted double ture DJR-MCPs, the majority of these viruses also encode for jelly-roll domain. Structure-guided phylogeny of MCP suggests that additional single jelly-roll minor capsid proteins (e.g., penton Yaravirus groups together with the MCPs of Pleurochrysis endemic proteins) and genome packaging ATPases of the FtsK-HerA su- viruses. Yaravirus expands our knowledge of the diversity of DNA perfamily. viruses. The phylogenetic distance between Yaravirus and all other According to this proposal, the evolutionary conservation of viruses highlights our still preliminary assessment of the genomic the three genes of the morphogenetic module in the DJR-MCP diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists. Significance Yaravirus | ORFan | NCLDV | metagenomics | capsid Most of the known viruses of amoeba have been seen to share iral evolution and classification have been subjects of an some features that eventually prompted authors to classify Vintense debate, especially after the discovery of giant viruses them into common evolutionary groups. Here we describe that infect protists (1–4). These viruses are predominantly Yaravirus, an entity that could represent either the first iso- characterized by the large size of their virions and genomes lated virus of Acanthamoeba spp. out of the group of NCLDVs or, in an alternative scenario, a distant and extremely reduced encoding hundreds to thousands of proteins, of which a large virus of this group. Contrary to what is observed in other iso- proportion currently remain without homologs in public se- lated viruses of amoeba, Yaravirus does not have a large/giant quence databases (5–10). These coding sequences are commonly particleoracomplexgenome,butatthesametimecarriesa referred as ORFans, and due to the lack of phylogenetic in- number of previously undescribed genes, including one encoding formation, their origin and function still represent a mystery – a divergent major capsid protein. Metagenomic approaches also (11 14). Strikingly, the increasing number of available viral ge- testified for the rarity of Yaravirus in the environment. nomes demonstrates that there is a huge set and great diversity of genes without homologs in current databases, which needs to Author contributions: F.S., P.D., E.C., P.C., A.L., B.L.S., and J.S.A. designed research; be further explored (11). Importantly, many amoebal virus P.V.M.B., G.P.O., T.B.M., A.C.S.P.A., J.-P.B., T.K., F.S., S.A., P.D., P.C., A.L., B.L.S., and ORFan genes have already been proven to be functional, being J.S.A. performed research; E.C., B.L.S., and J.S.A. contributed new reagents/analytic tools; P.V.M.B., G.P.O., J.-P.B., T.K., F.S., S.A., P.D., E.C., P.C., A.L., B.L.S., and J.S.A. analyzed data; expressed and encoding for components of the viral particles (6, and P.V.M.B., G.P.O., T.K., F.S., P.C., A.L., B.L.S., and J.S.A. wrote the paper. 15). However, the large set of ORFans makes it difficult to The authors declare no competing interest. predict the biology of viruses discovered through cultivation- This article is a PNAS Direct Submission. independent methods, such as metagenomics analysis, reinforc- Published under the PNAS license. ing the need for the complementary isolation and experimental Data deposition: Yaravirus genome data have been deposited at GenBank (https://www. characterization of new viruses. All currently known isolated ncbi.nlm.nih.gov/genbank/) under accession number MT293574. amoebal viruses are related to nucleocytoplasmic large DNA 1P.V.M.B. and G.P.O. contributed equally to this work. viruses (NCLDVs) (16). This group comprises families of eukaryotic 2To whom correspondence may be addressed. Email: [email protected] or viruses (Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodna- [email protected]. viridae, Marseilleviridae, and Mimiviridae) as well as other amoebal This article contains supporting information online at https://www.pnas.org/lookup/suppl/ virus lineages including pithoviruses, pandoraviruses, molliviruses, doi:10.1073/pnas.2001637117/-/DCSupplemental. medusaviruses, pacmanviruses, faustoviruses, klosneuviruses, and www.pnas.org/cgi/doi/10.1073/pnas.2001637117 PNAS Latest Articles | 1of8 Downloaded by guest on September 25, 2021 supermodule, to the exclusion of all other viruses, justifies the plasma membrane, suggesting the participation of a host re- establishment of a realm named Varidnaviria (21). Although ceptor in order to internalize the virions (Fig. 1B, SI Appendix, some NCLDVs, as pandoraviruses, seem to have lost the DJR- Fig. S2, blue arrows, and Movie S1). The replication cycle is then MCP gene, their genome harbors a large set of genes that sup- followed by the incorporation of individual or grouped Yaravirus port their classification into NCLDVs (and Varidnaviria, conse- particles inside endocytic vesicles, which, in a later stage of in- quently). Here we describe the discovery of Yaravirus, an fection, are found next to a region occupied by the nucleus amoeba virus with a puzzling origin and evolution. This virus has (Fig. 1 B–D and SI Appendix, Fig. S2, red arrows). The viral a genome that mainly consists of a near full set of genes that are factory then takes place and completely develops into its mature ORFans. Yaravirus could represent either the first isolated virus form, replacing the region formerly occupied by the cell nucleus of Acanthamoeba spp. out of the group of NCLDVs, inaugu- and recruiting mitochondria around its boundaries, likely to rating a group belonging to Varidnaviria, or, in an alternative optimize the availability of energy to construct the virions evolutive scenario, a distant and extremely reduced virus of this (Fig. 1E). The step corresponding to viral morphogenesis hap- group. Viral particles have a size of 80 nm, escaping the concept pens similarly as how it is observed for other viruses of amoeba. of large and giant viruses. Thus, Yaravirus expands our knowl- First, it starts by the appearance of small crescents in the edge about viral diversity and evolution. electron-lucent region of the factory (Fig. 1E and 2A). Next, step by step, the virions gain an icosahedral symmetry by the se- Results quential addition of more than one layer of protein or mem- Yaravirus Isolation and Replication Cycle. A prospecting study was branous components around its structure (Fig. 2A). The conducted by collecting samples of muddy water from creeks of constructed virions, with a capsid still empty, start then to mi-